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Glucose 6 phosphate dehydrogenase mutant

A phosphate dehydrogenase and mutant technology, applied in the directions of enzymes, oxidoreductases, enzymes, etc., can solve the problems of poor label stability, poor stability, shortened validity period of the kit, etc., and achieve good thermal stability and inhibition. The effect of high rate and specific enzyme activity

Active Publication Date: 2016-12-07
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Even so, the stability of kits based on EMIT technology is often not ideal, and the validity period of liquid reagents is not long, which is related to the stability of the labeled glucose-6-phosphate dehydrogenase
It is precisely due to the poor stability of glucose 6 phosphate dehydrogenase, coupled with the influence of small molecule markers, that the stability of the marker is even worse, resulting in a shortened validity period of the kit

Method used

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  • Glucose 6 phosphate dehydrogenase mutant
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Mutation library construction

[0037] 1. For the sequence shown in Sequence 2, use the method of total gene synthesis to synthesize (remove the stop codon), and clone it into the pET-22b vector, and the restriction sites used are NdeI and XhoI. The plasmid pET-G6PD thus obtained was used as a template for the following error-prone PCR and WHOP-PCR.

[0038] 2. Error-prone PCR reaction system and conditions

[0039] Table 1 The reaction system is 100ul:

[0040] name

Volume (ul)

dNTP Mixture (2.5mM each)

8

dTTP (100mM)

0.8

dCTP (100mM)

0.8

10*PCR Buffer

10

Upstream primer (5mM), SEQ ID NO: 3

20

Downstream primer (5mM), SEQ ID NO: 4

20

MnCl 2 (5mM)

10

Mg 2+ (25mM)

14

Taq enzyme (5U / ul)

1

Template (10ng / ul)

5

water

12

[0041] The reaction conditions are:

[0042] 95°C for 5min; 94°C for 30sec; 55°C for 30sec; 72°C for 2min; 30 cycles; 72°...

Embodiment 2

[0055] Screening of mutant libraries

[0056] 1. Culture, induction and expression

[0057] After mixing the obtained clones with a coating rod, they were collected in a centrifuge tube, and the plasmids were extracted, and the obtained plasmids were transformed into BL21(ED3) for the next step of screening. Next, the above-mentioned transformant was inoculated in a 96-well plate, wherein the last well was inoculated with the wild-type strain as a control, and the medium used was 150ul LB / well containing ampicillin antibiotic, and the orifice plate was used as a retention plate. The next day, transfer to another 96-well plate in the same order. The medium used is 150ul LB / well, and ampicillin antibiotics and IPTG are added for induction at the same time. Cultivate at 37°C for 6 hours, centrifuge at 3800rpm to collect bacteria, and remove the medium , the orifice plate is used as the analysis plate.

[0058] 2. Screening

[0059] Add 150ul lysis solution (100mM Tris, pH8.0; ...

Embodiment 3

[0062] Enzyme Activity Determination and Thermal Stability Analysis of Glucose 6-Phosphate Dehydrogenase

[0063] The purified glucose-6-phosphate dehydrogenase was diluted to about 0.1 ug / ml in 100 mM Tris, pH 8.0 buffer.

[0064] Take 50ul of the glucose 6-phosphate dehydrogenase in a 96-well PCR, select 8 gradients in the range of 40-50°C for heat treatment for 30min, and store at 4°C.

[0065] Transfer 50ul of heat-treated glucose 6-phosphate dehydrogenase to a 96-well plate, and at the same time take 50ul of unheated glucose 6-phosphate dehydrogenase on the same 96-well plate. Incubate at 37°C for 10 minutes.

[0066] Add the reaction solution (Tris, 100mM, pH7.4; glucose solution, 100mM; NADP, 5mM; ATP, 5mM; MgCl 2 , 5mM; hexokinase, 10KU / L; BSA, 0.05mg / ml), react at 37°C for 30min.

[0067] The absorbance at 340 nm was recorded using a microplate reader.

[0068]Divide the absorbance value of the heat-treated glucose 6-phosphate dehydrogenase by the absorbance value...

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Abstract

The invention relates to a glucose 6 phosphate dehydrogenase mutant. The mutant is obtained by mutating 44th-site glycine into valine and 319th-site glycine into valine in a glucose 6 phosphate dehydrogenase of which the amino acid sequence is disclosed as SEQ ID No.1; and compared with the glucose 6 phosphate dehydrogenase of which the amino acid sequence is disclosed as SEQ ID No.1 before mutation, the mutant has higher heat stability. The glucose 6 phosphate dehydrogenase mutant has the advantages of high heat stability and high inhibition rate; and the mutant enzyme can be used as a raw material for multiple small molecule detection kits, so that the obtained kit has higher stability and sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically provides a glucose 6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase) mutant with high thermal stability and high inhibition rate, and the mutant enzyme can be used as a diagnostic kit. important raw material. Background technique [0002] In the field of clinical testing, the detection methods of small molecule hormones and therapeutic drugs mainly include RIA, Elisa, chemiluminescence, mass spectrometry, homogeneous enzyme immunoassay, etc. Among them, RIA technology has been eliminated due to radioactive contamination; Elisa technology is more advanced than RIA technology and has no radioactive contamination. However, the whole process is in a heterogeneous mode, requiring multiple washings of the plate, and a round of detection takes at least 2 hours. , the report time is too long; chemiluminescence technology has the characteristics of high sensitivity, and the detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53G01N33/573
CPCC12N9/0006C12Y101/01049G01N33/573G01N2333/902
Inventor 邹炳德邹继华汪屹贾江花
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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